Mass spectrometers are widely used to analyze ions on the basis of their mass-to-charge ratio (m/z). Mass spectrometry has become a primary technique for analysis of proteins. More recently mass spectrometry has been applied to the analysis of large protein complexes. The development of electrospray ionization coupled to mass spectrometry has enabled the analysis of large intact protein complexes, even when the latter are held together by weak non-covalent interactions. The study of protein complexes is important in view of their role as a variety of functional modules in biological systems. A new field has thus emerged, termed native protein mass spectrometry, which focuses on analysis of such species at near-physiological conditions (i.e. at approximately neutral pH).
Typically, the large intact complex ions produced at native conditions have a relatively high mass and relatively low charge state and thus high m/z (typically exceeding m/z 5,000, or exceeding m/z 10,000). Hence, for the mass analysis of the large intact complex ions themselves, it has become a typical application for time-of-flight (TOF) mass analyzers due to their ability to access very high m/z, frequently coupled with dedicated quadrupole mass filters (operating at very low frequencies to extend the mass range). However, recently, electrostatic mass analyzers such as an ORBITRAP mass analyzer have also been employed for native protein complexes (US-2014-0027629-A1) with advantages in mass resolution.
However, for a thorough analysis and identification of the monomer structure of protein complexes, tandem or MSn mass spectrometry needs to be applied. Numerous approaches to dissociation of intact protein complexes have been described in the prior art, including Collision Induced Dissociation (CID), Electron Capture Dissociation (ECD) and Surface Induced Dissociation (SID). Much of the prior art in this area has been summarised and discussed recently in Belov, M. E.; Damoc, E.; Denisov, E.; Compton, P, D,; Makarov, A, A,; Kelleher, N, L. Anal. Chem., 2013, 85, 11163-11173. In that paper it has been shown that some relatively small protein complexes can be successfully dissociated into the constituent monomer subunits, which then, in turn, are preselected and fragmented in a Higher-Energy Collision Dissociation Cell (HCD cell). The approach relies on dissociation of the native protein complexes in a ‘fly-through’ mode between a source comprising a dual ion funnel interface with injection flatapole, a mass selector and a HCD cell of an ORBITRAP™ mass spectrometer. That approach, however, has been found to be unreliable for some large complexes such as GroEL native complexes and has been found to be inapplicable to large heteromeric complexes (e.g., GroEL-GroES 14:7 complex).
In another prior art approach, the activation of the native protein complexes in the skimmer region of an ion mobility/time-of-flight mass spectrometer (IMS-TOFMS) has been investigated (Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Evidence of macromolecular protein rings in the absence of bulk water. Science, 2005, 310, 1658-1661; and Benesch, J. L. P. Collisional activation of protein complexes: picking up the pieces. J. Am. Soc. Mass Spectrom., 2009, 20, 341-348). The restructuring and unfolding of the protein complexes of interest was reported as confirmed by IMS measurements. However, no dissociation of native protein complexes (i.e., ejection of the monomer subunits) was observed, probably due to the elevated pressure in the skimmer interface.
It is therefore desirable to provide a more effective method and apparatus for the fragmentation of a wider range of large protein complexes.
In view of the above background, the present invention has been made.